The present invention relates generally to isolated and purified proteins and nucleic acids which modulate the biological activity of methionine adenosyltransferase II (MAT II). More particularly, the present invention relates to isolated and purified MAT II xcex2 subunit polypeptides and isolated and purified nucleic acids encoding the same.
Methionine adenosyltransferase (MAT; S-adenosyl-L-methionine (AdoMet) synthetase, EC 2.5.1.6) is an essential enzyme that catalyzes the synthesis of S-adenosylmethionine (AdoMet) from L-methionine (L-Met) and ATP (Cantoni, G. L. (1953) J. Biol. Chem. 204: 403-416; Mudd, S. H. (1973) The Adenosyltransferases, Third Edition Ed. The Enzymes, Group Transfer (Part A) (Bayer, P. D., Ed.), III). AdoMet is the major methyl group donor, participating in the methylation of proteins, DNA, RNA, phospholipids and other small molecules (reviewed in Finkelstein et al. (1975) Biochem. Biophys. Res. Commun. 66: 81-7; Tabor, C. W., and Tabor, H. (1984) Adv. Enzymol. Relat. Areas Mol. Biol. 56: 251-82; Mudd et al. (1995) Disorders of transsulfuration, 7th Ed. The Molecular and Metabolic Basis of Inherited Diseases (Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D., Eds.), McGraw-Hill Inc., New York). In addition, AdoMet is the ultimate source of the propylamine moiety used in polyamine biosynthesis, and it serves as co-factor for other key enzymes in the one-carbon metabolism pathway (Finkelstein et al. (1975) Biochem. Biophys. Res. Commun. 66: 81-7; Tabor, C. W., and Tabor, H. (1984) Adv. Enzymol. Relat. Areas Mol. Biol. 56: 251-82; Mudd et al. (1995) Disorders of transsulfuration, 7th Ed. The Molecular and Metabolic Basis of Inherited Diseases (Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D., Eds.), McGraw-Hill Inc., New York). MAT is present in all living species, including thermophilic archaebacteria, plants, yeast, and mammals (reviewed in Tabor, C. W., and Tabor, H. (1984) Adv. Enzymol. Relat. Areas Mol. Biol. 56: 251-82; Kotb, M., and Geller, A. M. (1993) Pharmacol. Ther. 59: 125-43; Chiang, et al. (1996) FASEB J. 10:471-80; Mato et al. (1997) Pharmacol. Ther. 73: 265-80). Interestingly, most species have more than one MAT isozyme (Kotb, M., and Geller, A. M. (1993) Pharmacol. Ther. 59: 125-43).
Mammalian MAT exists in multiple forms that differ in their physical and kinetic properties among distinct species and even among different tissues of the same species. In mammals there are three forms designated MAT I, II, and III that differ in their tissue distribution and kinetic properties (Hoffman, J. L. (1983) Methods Enzymol. 94, 223-8; Mato et al. (1994) Adv. Exp. Med. Biol. 368, 113-7; Okada et al. (1981) Biochemistry 20, 934-40; Kotb et al. (1997) Trends Genet. 13, 51-2). MAT I and III are referred to as the hepatic forms because their expression is confined to the liver. By contrast, MAT II is found in all mammalian tissues that have been examined to date, including erythrocytes, lymphocytes, brain, kidney, testis, and liver (Okada et al. (1981) Biochemistry 20, 934-40; Oden, K., and Clarke, S. (1983) Biochemistry 22, 2978-2986; Kotb, M., and Kredich, N. M. (1985) J. Biol. Chem. 260, 3923-30; Langkamp-Henken et al. (1994) Biochim. Biophys. Acta 1201, 397-404; Liau et al. (1979) Cancer Res. 39, 162-69; Sullivan, D. M., and Hoffman, J. L. (1983) Biochemistry 22, 1636-41; Mitsui et al. (1988) J. Biol. Chem. 263, 11211-16; Horikawa et al. (1990) J. Biol. Chem. 265, 13683-86).
MAT I is a tetramer and MAT III is a dimer of an identical catalytic subunit, xcex11, encoded by the MATIA gene (Hoffman, J. L. (1983) Methods Enzymol. 94, 223-8; Horikawa, S., and Tsukada, K. (1991) Biochem. Int. 25, 81-90; Alvarez et al. (1993) Biochem. J. 293, 481-86; Sakata et al. (1993) J. Biol. Chem. 268, 13978-86; Ubagai et al. (1995) J. Clin. Invest. 96, 1943-47). On the other hand MAT II from leukemic T cells or from activated human lymphocytes is a hetero-oligmer consisting of xcex12 (53 kDa), xcex1xe2x80x22 (51 kDa) and xcex2 (38 kDa) subunits (Kotb, M., and Kredich, N. M. (1985) J. Biol. Chem. 260, 3923-30). The xcex12 and xcex1xe2x80x22 are the catalytic subunits while xcex2 appeared to have a regulatory function (De La Rosa et al. (1992) J. Biol. Chem. 267, 10699-704; De La Rosa et al. (1995) J. Biol. Chem. 270:21860-68; LeGros et al. (1997) J. Biol. Chem. 272, 16040-47; LeGros et al. (1999) Submitted). The xcex12 and xcex1xe2x80x22 subunits are immunologically crossreactive and essentially identical to each other, but quite different from the xcex2 subunit. The xcex12 subunit, which appears to be posttranslationally processed to yield xcex1xe2x80x22 (Kotb, M., and Kredich, N. M. (1985) J. Biol. Chem. 260, 3923-30), is encoded by the MAT2A gene which is homologous, but different from MAT1A gene (Kotb et al. (1997) Trends Genet. 13, 51-2; Horikawa, S., and Tsukada, K. (1991) Biochem. Int. 25, 81-90; De La Rosa et al. (1995) J. Biol. Chem. 270:21860-68).
The human MAT II from human lymphocytes has been analyzed to a certain extent (Kotb, M., and Kredich, N. M. (1985) J. Biol. Chem. 260: 3923-30; Kotb, M., and Kredich, N. M. (1990) Biochim. Biophys. Acta 1039(2): 253-60; De La Rosa et al. (1992) J. Biol. Chem. 267: 10699-704; De La Rosa et al. (1995) J. Biol. Chem. 270, 21860-8; LeGros et al. (1997) J. Biol. Chem. 272, 16040-7), and it has been shown that the form present in activated lymphocytes consists of distinct subunits (Kotb, M., and Kredich, N. M. (1985) J. Biol. Chem. 260, 3923-30; De La Rosa et al. (1992) J. Biol. Chem. 267, 10699-704). The catalytic MAT II xcex12 subunit, which is encoded by the MAT2A gene, was cloned and characterized and found to be homologous, but different from the catalytic xcex11 subunit of the liver MAT I/III isozyme (Horikawa, S., and Tsukada, K. (1991) Biochem. Int. 25, 81-90; Alvarez et al. (1993) Biochem. J. 293, 481-6; Sakata et al. (1993) J. Biol. Chem. 268, 13978-86; Horikawa et al. (1990) J. Biol. Chem. 265, 13683-6; De La Rosa et al. (1995) J. Biol. Chem. 270, 21860-8). The MAT II xcex12 subunit, which has a calculated molecular weight of 43,600, migrates on SDS-PAGE gels as a 53 kDa protein, and is postranslationally modified to generate MAT II xcex12xe2x80x2 subunit (Kotb, M., and Kredich, N. M. (1985) J. Biol. Chem. 260, 3923-30). The catalytic xcex12/xcex12xe2x80x2 subunits are found in native MAT II associated with a catalytically inactive subunit designated MAT II xcex2, which migrates on SDS-PAGE as a 38-kDa protein (Kotb, M., and Kredich, N. M. (1985) J. Biol. Chem. 260, 3923-30; De La Rosa et al. (1992) J. Biol. Chem. 267, 10699-704; LeGros et al. (1997) J. Biol. Chem. 272, 16040-7).
Earlier work has shown that physiological activation of human lymphocytes induces downregulation of the xcex2 subunit with co-incidental alterations in MAT II kinetic properties (LeGros et al. (1997) J. Biol. Chem. 272, 16040-47). However, this differential expression of the xcex2 subunit has not been fully characterized. Further characterization of the MAT II xcex2 subunit, including the regulatory role of the MAT II xcex2 subunit, thus represents an ongoing need in the art.
The present invention discloses isolated and purified nucleic acids encoding the subunit of methionine adenosyltransferase II (MAT II), to isolated and purified MAT II xcex2 subunit polypeptides, and to the characterization of the role played by the MAT II xcex2 subunit in modulating the biological activity of MAT II. More preferably, a polypeptide of the invention is a recombinant polypeptide. Even more preferably, a polypeptide of the present invention comprises a vertebrate MAT II xcex2 subunit polypeptide. Even more preferably, a polypeptide of the present invention comprises a mammalian MAT II xcex2 subunit polypeptide. Even more preferably, a polypeptide of the present invention comprises a human MAT II xcex2 subunit polypeptide. Even more preferably, a polypeptide of the present invention comprises an amino acid sequence from the amino acid residue sequences of any of FIGS. 1-5 (corresponding to SEQ ID NOs: 17, 19, 21, 23 and 25, respectively).
The present invention also provides an isolated and purified polynucleotide that encodes a MAT II xcex2 subunit polypeptide that modulates the biological activity of MAT II. In a preferred embodiment, a polynucleotide of the present invention comprises a DNA molecule from a vertebrate species. A preferred vertebrate is a mammal. A preferred mammal is a human. More preferably, a polynucleotide of the present invention encodes a polypeptide comprising an amino acid residue sequence of any of FIGS. 1-5 (corresponding to SEQ ID NOs: 17, 19, 21, 23 and 25, respectively). Most preferably, an isolated and purified polynucleotide of the invention comprises a nucleotide base sequence of any of FIGS. 1-5.
In another embodiment, the present invention provides an antibody immunoreactive with a MAT II xcex2 subunit polypeptide as described above. FIGS. 1-5 (SEQ ID NOs: 16-25) set forth nucleotide and amino acid sequences from an exemplary vertebrates, human. Also provided by the present invention are antibodies immunoreactive with homologues or biologically equivalent MAT II xcex2 subunit polynucleotides and polypeptides found in other vertebrates. Preferably, an antibody of the invention is a monoclonal antibody. More preferably, the MAT II xcex2 subunit polypeptide comprises a human MAT II xcex2 subunit polypeptide. Even more preferably, the MAT II xcex2 subunit polypeptide comprises an amino acid residue sequence of any of FIGS. 1-5 (corresponding to SEQ ID NOs: 17, 19, 21, 23 and 25, respectively).
In another aspect, the present invention provides a process of producing an antibody immunoreactive with a MAT II xcex2 subunit polypeptide as described above, the process comprising the steps of (a) transfecting a recombinant host cell with a polynucleotide that encodes a biologically active MAT II xcex2 subunit polypeptide; (b) culturing the host cell under conditions sufficient for expression of the polypeptide; (c) recovering the polypeptide; and (d) preparing the antibody to the polypeptide. FIGS. 1-5 (SEQ ID NOs: 16-25) set forth nucleotide and amino acid sequences from an exemplary vertebrate, human. Preferably, the host cell is transfected with a polynucleotide of any of FIGS. 1-5 (SEQ ID NOs: 16, 18, 20, 22, 24, respectively). Even more preferably, the present invention provides an antibody prepared according to the process described above. Also provided by the present invention is the use of homologues or biologically equivalent polynucleotides and polypeptides found in other vertebrates to produce antibodies.
Alternatively, the present invention provides a process of detecting a MAT II xcex2 subunit polypeptide as described above, wherein the process comprises immunoreacting the polypeptide with an antibody prepared according to the process described above to form an antibody-polypeptide conjugate, and detecting the conjugate.
In yet another embodiment, the present invention provides a process of detecting a messenger RNA transcript that encodes a MAT II xcex2 subunit polypeptide as described above, wherein the process comprises hybridizing the messenger RNA transcript with a polynucleotide sequence that encodes that polypeptide to form a duplex; and detecting the duplex. Alternatively, the present invention provides a process of detecting a DNA molecule that encodes a MAT II xcex2 subunit polypeptide as described above, wherein the process comprises hybridizing DNA molecules with a polynucleotide that encodes a biologically active MAT II xcex2 subunit polypeptide to form a duplex; and detecting the duplex.
In another aspect, the present invention provides an assay kit for detecting the presence of a MAT II xcex2 subunit polypeptide in a biological sample, where the kit comprises a first container containing a first antibody capable of immunoreacting with a biologically active MAT II xcex2 subunit polypeptide, with the first antibody. Preferably, the first antibody is present in an amount sufficient to perform at least one assay. Also preferably, an assay kit of the invention further comprises a second container containing a second antibody that immunoreacts with the first antibody. More preferably, the antibodies used in an assay kit of the present invention are monoclonal antibodies. Even more preferably, the first antibody is affixed to a solid support. More preferably still, the first and second antibodies comprise an indicator, and, preferably, the indicator is a radioactive label, a fluorescent label or an enzyme.
In an alternative aspect, the present invention provides an assay kit for detecting the presence, in biological samples, of a MAT II xcex2 subunit polypeptide, the kits comprising a first container that contains a second polynucleotide identical or complementary to a segment of at least 10 contiguous nucleotide bases of a polynucleotide that encodes a biologically active MAT II xcex2 subunit polypeptide.
In another embodiment, the present invention provides an assay kit for detecting the presence, in a biological sample, of an antibody immunoreactive with a MAT II xcex2 subunit polypeptide, the kit comprising a first container containing a biologically active MAT II xcex2 subunit polypeptide that immunoreacts with the antibody, with the polypeptide present in an amount sufficient to perform at least one assay.
In still a further embodiment, this invention pertains to therapeutic methods based upon the modulation of the biological activity of MAT II via MAT II xcex2 subunit polynucleotides and polypeptides as described herein. Such therapeutic methods include administration of a soluble form of the MAT II xcex2 subunit polypeptide as well as gene therapy approaches using an isolated and purified polynucleotide of the present invention.
Thus, a key aspect of this invention pertains to the discovery of the novel MAT II xcex2 subunit polypeptides and nucleic acids. Preferred nucleic acid and amino acid sequences are described in FIGS. 1-5 (SEQ ID NOs: 16-25).
It is thus another aspect of this invention to provide a purified and isolated MAT II xcex2 subunit polypeptide having a role in the biological activity of MAT II.
The foregoing aspects and embodiments have broad utility given the biological significance of the MAT II enzyme. By way of example, the foregoing aspects and embodiments are useful in the preparation of screening assays and assay kits that are used to identify compounds that affect or modulate MAT II biological activity, or that are used to detect the presence of the proteins and nucleic acids of this invention in biological samples. Additionally, it is well known that isolated and purified polypeptides have utility as feed additives for livestock and further polynucleotides encoding the polypeptides are thus useful in producing the polypeptides.
Some of the aspects and objects of the invention having been stated hereinabove, other aspects and objects will become evident as the description proceeds, when taken in connection with the accompanying drawings and examples as best described hereinbelow.